The Streptococci make up a medically important genera of microbes known to cause several types of disease in humans, including, for example, otitis media, conjunctivitis, pneumonia, bacteremia, meningitis, sinusitis, pleural empyema and endocarditis, and most particularly meningitis, such as for example infection of cerebrospinal fluid. Since its isolation more than 100 years ago, Streptococcus pneumoniae has been one of the more intensively studied microbes. For example, much of our early understanding that DNA is, in fact, the genetic material was predicated on the work of Griffith and of Avery, Macleod and McCarty using this microbe. Despite the vast amount of research with S. pneumoniae, many questions concerning the virulence of this microbe remain. It is particularly preferred to employ Streptococcal genes and gene products as targets for the development of antibiotics.
The frequency of Streptococcus pneumoniae infections has risen dramatically in the past few decades. This has been attributed to the emergence of multiply antibiotic resistant strains and an increasing population of people with weakened immune systems. It is no longer uncommon to isolate Streptococcus pneumoniae strains which are resistant to some or all of the standard antibiotics. This phenomenon has created an unmet medical need and demand for new anti-microbial agents, vaccines, drug screening methods, and diagnostic tests for this organism.
Moreover, the drug discovery process is currently undergoing a fundamental revolution as it embraces "functional genomics," that is, high throughput genome- or gene-based biology. This approach is rapidly superseding earlier approaches based on "positional cloning" and other methods. Functional genomics relies heavily on the various tools of bioinformatics to identify gene sequences of potential interest from the many molecular biology databases now available as well as from other sources. There is a continuing and significant need to identify and characterize further genes and other polynucleotides sequences and their related polypeptides, as targets for drug discovery. The enzyme UDP-N-acetylenolpyruvylglucosamine reductase, encoded by the gene MurB catalyses the reduction of UDP-N-acetylpyruvylglucosamine to UDP-N-acetyl muramate, with the concomitant oxidation of NADPH. N-acetyl muramate is a precusor for peptidoglycan biosynthesis.
The gene has been sequenced from Escherichia coli (Pucci, M. J., Discotto, L. F. & Dougherty T. J., 1992, J. Bacteriol., 174, 1690-1693) and the enzyme over-expressed, purified and kinetically characterized (Benson, T. E., Marquardt, J. L., Marquardt, A. C., Etzkom, F. A. & Walsh, C. T. (1993) Biochemistry, 32, 2024-2030). There is also a crystal structure of this enzyme (Benson, T. E., Walsh, C. T., & Hogle, J. M., (1996) Structure, 4, 47-54).
The gene has also been sequenced from Bacillus subtilis and shown to be essential in this organism (Rowland et al., (1995) Gene 164, 113-116).
The discovery of a MurB homologue in the human pathogen Streptococcus pneumoniae will allow us to produce UDP-N-acetylenolpyruvylglucosamine reductase enzyme which can then be used to screen for novel antibiotics. Inhibitors of this enzyme will have utility in anti-bacterial therapy as they will prevent the construction of the bacterial cell wall.
Clearly, there exists a need for polynucleotides and polypeptides, such as the MurB embodiments of the invention, that have a present benefit of, among other things, being useful to screen compounds for antibiotic activity. Such factors are also useful to determine their role in pathogenesis of infection, dysfunction and disease. There is also a need for identification and characterization of such factors and their antagonists and agonists to find ways to prevent, ameliorate or correct such infection, dysfunction and disease.
Certain of the polypeptides of the invention possess significant amino acid sequence homology to a known MurB from B. subtilis protein.